Tumor-specific efficacy of transforming growth factor-beta RI inhibition in Eker rats.

PURPOSE: Transforming growth factor beta (TGF-beta), which generally stimulates the growth of mesenchymally derived cells but inhibits the growth of epithelial cells, has been proposed as a possible target for cancer therapy. However, concerns have been raised that whereas inhibition of TGF-beta signaling could be efficacious for lesions in which TGF-beta promotes tumor development and/or progression, systemic pharmacologic blockade of this signaling pathway could also promote the growth of epithelial lesions. EXPERIMENTAL DESIGN: We examined the effect of a TGF-beta inhibitor on mesenchymal (leiomyoma) and epithelial (renal cell carcinoma) tumors in Eker rats, which are genetically predisposed to develop these tumors with a high frequency. RESULTS: Blockade of TGF-beta signaling with the ALK5/type I TGF-beta R kinase inhibitor, SB-525334, was efficacious for uterine leiomyoma; significantly decreasing tumor incidence and multiplicity, and reducing the size of these mesenchymal tumors. However, SB-525334 was also mitogenic and antiapoptotic for epithelial cells in the kidney and exacerbated the growth of epithelial lesions present in the kidneys of these animals. CONCLUSION: Although pharmacologic inhibition of TGF-beta signaling with SB-525334 may be efficacious for mesenchymal tumors, inhibition of this signaling pathway seems to promote the development of epithelial tumors.     

Shuttle vectors for <Emphasis Type="Italic">Candida albicans</Emphasis>: control of plasmid copy number and elevated expression of cloned genes

Plasmids containing the inosine monophosphate dehydrogenase gene CaIMH3 from Candida albicans strain ATCC 32354 transform their host to resistance against mycophenolic acid (MPA). The transformants maintain the plasmids at a high copy number (20–40 per cell) and express the CaIMH3 gene at very high levels relative to untransformed controls. The plasmid copy number can be controlled by the concentration of MPA in the media. The transformation procedure is reproducible and the efficiency of transformation is high, up to 15,000 per microgram. Unrearranged plasmids are readily recovered by transforming total DNA from transformants back into Escherichia coli. C. albicans genes cloned into the plasmid are expressed at elevated levels relative to untransformed controls. A derivative vector containing the CaMAL2 promoter and termination sequences expresses the CaERG11 ORF at high levels and confers moderate resistance to fluconazole. These shuttle vectors should facilitate global genomics approaches in C. albicans that have been hampered by its diploid genome.     

Cell-specific coupling of the cloned human 5-HT1F receptor to multiple signal transduction pathways

We recently described the cloning of a fifth member of the 5-hydroxytryptamine (5-HT)₁ (serotonin₁) receptor class that inhibits adenylyl cyclase, namely the human 5-HT_1F receptor (Adham et al. 1993 a). In the present study we have examined in greater detail the functional coupling of the 5-HT_1F receptor in two different cell lines, NIH-3T3 and LM(tk^–) fibroblasts (receptor densities of 1.7 and 4.4 pmol/mg protein, respectively). The maximal inhibitory response elicited by 5-HT was significantly greater in NIH-3T3 as compared to LM(tk^–) cells, whereas the EC₅₀ values were comparable.To investigate the relationship between receptor occupancy and inhibition of cAMP accumulation mediated by 5-HT_1F receptors in NIH-3T3 cells (and hence the degree of receptor reserve), we used the irreversible receptor antagonist N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ). The half-maximal response required only about 10% receptor occupancy, consistent with a receptor reserve of 90% (88±2.1%, n = 4) for 5-HT-induced inhibition of FSCA. Despite the presence of such a high degree of receptor reserve, a range of intrinsic activities was displayed by structurally diverse classes of compounds. For example, sumatriptan and lysergol were as efficacious as 5-HT itself and thus acted as full agonists, whereas metergoline and 1-NP behaved as partial agonists and as shown previously (Adham et al. 1993a), methiothepin was a silent antagonist (K_b = 438 nM).We have also investigated activation of additional signal transduction pathways by the 5-HT_1F receptor and found that the responses differ in the two cell lines with respect to stimulation of phospholipase C. For example, in NIH-3T3 cells no elevation of inositol phosphates (IP) of [Ca²⁺]_i was observed even at very high agonist concentrations (100 M). In contrast, in LM(tk^–) cells concentrations of 5-HT as low as 10 nM induced stimulation of IP and a rapid increase of [Ca²⁺]i. The 5-HT_1F receptor failed to alter arachidonic acid release in either cell line.The maximal increase in IP accumulation in LM(tk^–) cells was modest, averaging about 100% above basal. The increases of IP and [Ca²⁺]_i required 5-HT concentrations less than one order of magnitude greater than those inhibiting FSCA (EC₅₀ = 17, 55 and 8 nM, respectively), and both responses were blocked by 100 M methiothepin. All three responses (cAMP, IP, and [Ca²⁺]_i) were sensitive to pertussis toxin pre-treatment, suggesting the involvement of G_i/G_o protein(s) in these signal transduction pathways. [Ca²⁺]_i was also elevated by sumatriptan, which may provide a mechanism by which this drug causes constriction of the vasculature. In conclusion, these data indicate that the human 5-HT_1F receptor can couple to multiple effectors, and that this coupling is cell-type dependent.Abbreviations FSCA forskolin-stimulated cAMP accumulation - [Ca²⁺] intracellular free calcium concentration - AA arachidonic acid - EEDQ N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline - CHO chinese hamster ovary cell - LM(tk^–) mouse fibroblast cell - B_max maximal binding site density - K_i apparent dissociation constant obtained from competition binding studies - G protein guanine nucleotide-binding protein - HBS HEPES-buffered saline - IP inositol phosphates - IP₃ inositol 1,4,5 trisphosphate - PLC phospholipase C - K_b antagonist dissociation constant - K_d equilibrium dissociation constant - N-1F-6 stable NIH-3T3 cells expressing the cloned 5-HT_1F receptor - L-1F-3 stable LM(tk^–) cells expressing the cloned 5-HT_1F receptor - PTX pertussis toxin - BSA bovine serum albumin - METH methiothepin - SUMA sumatriptan - 5-MeO-DMT 5-methoxy-N,N-dimethyltryptamine - 1-NP 1-(1-napthyl)piperazine - 5-CT 5-carboxyamidotryptamine Correspondence to: N. Adham at the above address     

Feasibility of use of magnesium reabsorbable scaffolds in complex coronary artery disease: True bifurcation,multiple overlapping scaffolds and chronic total occlusion treatment

We present first-in-human treatment with bioabsorbable magnesium scaffolds for percutaneous coronary intervention in a patient with nickel allergy. We present images from angiography and optical coherence tomography at three months. We also review the current status of these novel devices.     

Perinatal Exposure to Bisphenol A or Diethylstilbestrol Increases the Susceptibility to Develop Mammary Gland Lesions After Estrogen Replacement Therapy in Middle-Aged Rats

The development of the mammary gland is a hormone-regulated event. Several factors can dysregulate its growth and make the gland more susceptible to cellular transformation. Among these factors, perinatal exposure to xenoestrogens and hormone replacement therapy has been associated with increased risk of developing breast cancer. Here, we assessed the effects induced by estrogen replacement therapy (ERT) in ovariectomized (OVX) middle-aged rats and whether perinatal exposure to diethylstilbestrol (DES) or bisphenol A (BPA) modified these effects in the mammary gland. Pregnant rats were orally exposed to vehicle, 5 μg DES/kg/day, or 0.5 or 50 μg BPA/kg/day from gestational day 9 until weaning. Then, 12-month-old offspring were OVX and treated with 17β-estradiol for 3 months. Morphological changes and the percentage of epithelial cells that proliferated or expressed estrogen receptor alpha (ESR1) and progesterone receptor (PR) were analyzed in mammary gland samples of 15-month-old animals. ERT induced lobuloalveolar hyperplasia and ductal cysts in the mammary gland of middle-aged rats, associated with a higher proliferation index of epithelial cells. Perinatal exposure to DES followed by ERT increased the number of cysts and induced the formation of fibroadenoma and ductal carcinoma in situ, without modifying the expression of ESR1 or PR. Also, after 3 months of ERT, BPA-exposed rats had a higher incidence of ductal hyperplasia and atypical lobular hyperplasia than animals under ERT alone. In conclusion, perinatal exposure to xenoestrogens increases the susceptibility of the mammary gland to develop cysts and hyperplastic lesions when confronted with ERT later in life.